The ultraviolet difference spectra of horse liver alcohol dehydrogenase with coenzymes and substrate analogues bear a very close resemblance to those obtained by perturbation of the coenzymes and their analogues by acid, NaC1, dioxan or t-butanol. This indicates the composite ionic and nonpolar environment the coenzymes experience at the active site of the enzyme. The phosphorescence spectra do not indicate the presence of ionized tyrosine in ternary complexes involving enzyme, coenzyme and substrate analogues. The thermodynamic parameters characterizing self-association and ligand binding of several proteins were reviewed. Based on the thermochemical behavior of small molecule interactions, it is concluded that the strengthening of hydrogen bonds in the low dielectric protein interior and van der Waals interactions are the most important factors contributing to the observed negative values of delta H degree and delta S degree and hence to the stability of protein association complexes.